The main objective of this proposal is to elucidate the mechanisms by which the histone H2AX protein signals cellular responses to DNA double strand breaks (DSBs). DSBs are generated by genotoxic agents and are intermediates during physiologic processes such as DNA replication, meiosis and the generation (VDJ recombination) and diversification (class switch recombination) of lymphocyte antigen receptor genes. We have shown that H2AX deficient mice exhibit genomic instability and an increased predisposition cancer, including lymphomas clonal translocations, some of which involve antigen receptor loci. These data indicate that H2AX is involved in the repair of and/or cellular response to genotoxic and physiologic DSBs. H2AX is phosphorylated (generating ?-H2AX), acetylated, and ubiquitylated in chromatin around DSBs; however, the function(s) of ?-H2AX and these other modified forms of H2AX are not known. H2AX deficient mice are lymphopenic and 3-H2AX forms at antigen receptor loci undergoing V(D)J recombination, suggesting that H2AX functions in the repair of and/or cellular response to DSBs generated during lymphocyte antigen receptor gene assembly. We have shown that the kinase activity of ATM is required for the repair of DSBs generated during antigen receptor gene assembly and for the activation of a broadly functional genetic program in response to these physiologic DSBs. Since ATM is the major kinase that phosphorylates H2AX, our data raise the possibility that the ATM functions through the phosphorylation of ?-H2AX to mediate these processes. Within this application, we show that H2AX is not required for the repair of DSBs generated during antigen receptor gene assembly, but is required to activate gene expression in response to these physiologic DSBs. Thus, we hypothesize that H2AX is an important intermediate in activating transcriptional pathways in response to DSBs due, at least in part, to a requirement to generate 3-H2AX in chromatin around these DNA lesions. Here, we propose to use a novel cell line based approach whereby DSBs can be induced at precise locations in the genome and elucidate the cis-acting and trans-acting factors that regulate the formation of ?-H2AX at these breaks. In addition, we propose to elucidate the transcriptional pathways that are activated by ?-H2AX, determine how they are activated, and identify the target genes that are regulated by these pathways. PUBLIC HEALTH RELEVANCE: The histone H2AX protein is centrally important in the cellular responses to damaged DNA, which are critical for the repair of damaged DNA and for the elimination of cells with persistent damage since they are at risk for genomic instability. We have shown that H2AX functions as a critical signaling intermediate in the activation of gene expression changes in response to DNA double strand breaks. Here, we propose to elucidate the genetic program initiated by H2AX, identify the transcriptional pathways that are activated, and determine how they are activated by H2AX.